# Patent

## Title
Flavonoid-3′,5′-hydroxylase gene of commelina communis

## Abstract

To provide a highly expressed F3′5′H gene, whereby a blue moth orchid can be produced and a method using the gene for producing Orchids having blue flowers.
A gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which has an amino acid sequence depicted in SEQ ID No: 2 or an amino acid sequence having at least 90% of homology to the amino acid sequence depicted in SEQ ID No: 2. A method for producing an orchid having a blue flower, which comprises transfecting a white moth orchid with the gene, a gene encoding a dihydroflavonol 4-reductase of Torenia or Gerbera; a gene encoding a flavanone 3-hydroxylase; and a gene encoding an anthocyanidin synthase and expressing the genes.

## Background
TECHNICAL FIELD
The present invention relates to a method for producing a plant having a blue flower color by employing the gene recombination technology. Particularly, the present invention relates to a method for producing Orchids having a blue flower color by using a gene encoding a flavonoid-3′,5′-hydroxylase (F3′5′H) which is one of enzymes of Commelina Communis for synthesizing pigments.
BACKGROUND ART
The color of flower is a particularly important character in ornamental plants, and flowers having various colors have been produced by cross breeding heretofore. However, in the case of the cross breeding, gene sources are limited to species which are capable of cross breeding, and colors to be changed are limited. Further, in a case where only a specific character such as flower color is introduced into a specific variety, it is necessary to repeat backcrossing for long generations, and a lot of effort and time are required. Further, a period of cross breeding varies depending on plant species, and some plants take from a few years to a few decades for blossom. Particularly, Orchids such as moth orchid and cymbidium require a long time for blossom, and it takes a long time to develop such plants. Therefore, although demanded in markets, a superior new variety of moth orchid or cymbidium having a new flower color, particularly blue flower, has not been produced.
In recent years, it is possible to carry out cross breeding over species or genus by the recombinant DNA technology, and it is expected to produce a new variety having a color which cannot be obtained by the conventional cross breeding.
The color of flower derives mainly from three types of pigments: anthocyanin, carotenoid and betalain. Among them, anthocyanin (from orange to blue color) having the broadest maximum absorption wavelength has a role to govern blue color. Anthocyanin is one of flavonoids and biologically synthesized through a metabolic pathway shown in FIG. 1. The color of anthocyanin substantially depends on its chemical structure, and the more the number of hydroxyl groups in a benzene ring is, the more the color becomes blue. The hydroxylation of the benzene ring is catalyzed by a flavonoid 3′-hydroxylase (F3′H) and a flavonoid 3′,5′-hydroxylase (F3′5′H). In a case where there is neither F3′H activity nor F3′5′H activity in petal cells, pelargonidin (from orange color to red color) is synthesized, and in a case where there is F3′H activity, cyanidin (from red to crimson color) is synthesized. Further, in a case where there is F3′5′H activity, delphinidin (blue color) is synthesized. Therefore, in order to produce the blue flower color, the role of F3′5′H is considered to be important.
From such a viewpoint, a study is in progress to produce a plant having a blue flower by the gene recombination using F3′5′H.
As conventionally known genes encoding the F3′5′H, genes derived from plants such as Campanula medium, Catharanthus roseus, Petunia, Eustoma grandiflorum, Nierembergia sp., Verbena, Gentiana, Gossypium hirsutum, Lycianthes rantonnei, Solanum tuberosum and Torenia, have been known, however, a gene encoding the F3′5′H which is isolated from a Commelina communis has not been reported.
As examples wherein a flower color is changed by using a conventionally known gene, a method for producing a blue carnation by transfecting a carnation DFR (dihydroflavonol 4-reductase) deficient variety with a F3′5′H gene and DFR gene which are derived from Petunia (Patent Document 1) and a method for producing a blue rose by transfecting a rose of which internal metabolism pathway is suppressed, with a F3′5′H gene derived from Viola×wittrockiana (Patent Document 2) have been reported.
On the other hand, it has been reported to change the flower color of moth orchid by overexpressing an endogenous gene, however, a blue moth orchid has not been produced (Non-Patent Document 1). Further, it has not been reported to have produced a blue variety of cymbidium.

    Patent Document 1: WO1996/036716
    Patent Document 2: WO2005/017147
    Non-Patent Document 1: Su and Hsu, Biotechnology Letters (2003) 25: 1933-1939.


DISCLOSURE OF THE INVENTION
Object to be Accomplished by the Invention
In order to produce a blue flower, the F3′5′H is known to play an important role, and a F3′5′H gene which can be expressed more strongly has been desired.
It is an object of the present invention to find out a highly expressed type F3′5′H gene, whereby blue moth orchid can be produced, and to produce Orchids having a blue flower by using such a highly expressed type F3′5′H gene.
Means to Accomplish the Object
The present inventors have conducted an extensive study in order to accomplish the above object, and as a result they have found that a F3′5′H gene which is derived from Commelina communis has a higher effect than that of conventional genes, and that by transfecting moth orchid with the above gene, the color of its flower can be changed to blue. Thus, the present invention has been accomplished.
That is, the present invention relates to a gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which comprises an amino acid sequence depicted in SEQ ID No: 2 or an amino acid sequence having at least 90% of homology to the amino acid sequence depicted in SEQ ID No: 2.
Further, the present invention relates to a vector, which contains the above gene.
Further, the present invention relates to a method for producing a flower color-changed plant, which comprises transfecting a plant with the above gene and expressing the gene.
Further, the present invention relates to a method for producing a flower color-changed plant, which comprises transfecting an Orchid with the above gene and a gene encoding a dihydroflavonol 4-reductase of Torenia or Gerbera and expressing the genes.
Further, the present invention relates to a method for producing an Orchid having a blue flower, which comprises transfecting an Orchid having a white flower with the above gene; a gene encoding a dihydroflavonol 4-reductase of Torenia or Gerbera; a gene encoding a flavanone 3-hydroxylase; and a gene encoding an anthocyanidin synthase and expressing the genes.
Further, the present invention relates to a flower color-changed plant, which is produced by the above method, a progeny having the same characters as the changed flower color; or its tissue.
Further, in the present invention, “flavonoid 3′,5′-hydroxylase” (F3′5′H) is an enzyme which catalyzes a reaction to produce dihydromyricetin from dihydrokaempferol. Further, “flavanone 3-hydroxylase” (F3H) is an enzyme which catalyzes a reaction to produce dihydrokaempferol from naringenin. “Dihydroflavonol 4-reductase” (DFR) is an enzyme which catalyzes a reaction to produce leucodelphinidin from dihydromyricetin. “Anthocyanidin Synthase” (ANS) is an enzyme which catalyzes a reaction to produce delphinidin from leucodelphinidin.
Effect of the Present Invention
By using the F3′5′H gene derived from Commelina communis, plants having various blue flower colors can be produced. Particularly, by the present invention, it is possible to produce Orchids having blue flower colors which cannot be accomplished by the conventional cross breeding methods.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a synthetic pathway of anthocyanin.
FIG. 2 is a plasmid vector used for transfecting a petal with a gene. P-CaMV35S is a cauliflower mosaic virus 35S promoter. Ω is an omega sequence of tobacco mosaic virus. T is a transcription termination sequence derived from a cauliflower mosaic virus.
FIG. 3 is transfection of a petal of Phal. amabilis with a different ANS gene derived from a plant.
FIG. 4 is transfection of a petal of Phal. amabilis with a different DFR gene derived from a plant.
FIG. 5 is transfection of a petal of Phal. amabilis with a different F3′5′H gene derived from a plant.
FIG. 6 is an identification of a gene required for changing the flower color of a white moth orchid.
FIG. 7 is a schematic view (1) showing a procedure for construction of DNA for transformation of moth orchid.
FIG. 8 is a schematic view (2) of a procedure for construction of DNA for transformation of moth orchid.
FIG. 9 is transfection of a petal of a white cymbidium with genes.



BEST MODES FOR CARRYING OUT THE INVENTION
Now, the present invention will be described in detail, and by these descriptions, other objects, characteristics and superiorities of the present invention will become apparent for those skills in the art. Further, the descriptions of the present specification including the following explanations, specific examples, etc. show preferred embodiments, and those skilled in the art can make various changes and/or modifications (or alternations) of the invention within the concept and the scope of the present invention disclosed in the present specification. Further, all Patent Documents and references cited in the present specification are cited for the purpose of explanation, and it should be understood that their contents are included as a part of the present specification.
By paying attention to a F3′5′H gene which relates to a blue pigment (delphinidin), the present inventors have transfected moth orchid with a known F3′5′H gene which is derived from petunia and has shown practical results in carnation, in order to produce blue orchids. However, the desired effect cannot be obtained. Thus, the present inventors have carried out cloning of F3′5′H genes of various plants in order to find a superior gene. As a result, they have found that a gene (sequence number: 1) derived from Commelina communis has a remarkably high pigment production performance. In a case where a petal cell of moth orchid is transfected with the above gene, five times of delphinidin is produced, as compared to the gene derived from petunia which is the prior art. Further, in a case where a petal cell of a red moth orchid is transfected with the gene of the present invention, the flower color is changed to blue tone purple.
Further, it has been found that the flower color is changed from white to blue by transfecting a petal cell of a white moth orchid with the F3H gene which catalyzes an up stream reaction than the F3′5′H and a DFR gene and an ANS gene which catalyze a down stream reaction than the F3′5′H in the synthesis pathway of anthocyanin, in addition to the above F3′5′H gene. Particularly, in a case where a DFR gene derived from Gerbera or Torenia is used, a flower has a deeper blue color.
The gene derived from Commelina communis which is found in the present invention is superior to conventional genes, and by employing the gene of present invention, it is possible to produce a blue type moth orchid. Further, the superiority of the gene of the present invention has been proven on cymbidium as well as on moth orchid, and it has been found that the gene of the present invention can be applied for changing flower colors of various flowering plants.
The F3′5′H gene is isolated from the genome DNA of Asiatic dayflower (nomenclature: Commelina communis) or cDNA reversetranscribed from a mRNA transcribed from the above genome DNA. The F3′5′H gene of the present invention is a gene depicted in SEQ ID NO.: 1 encoding a protein depicted in SEQ ID NO.: 2. The gene of the present invention includes genes having a high homology to the above gene, so long as such genes have the F3′5′H enzyme activity. The high homology may be at least 85% homology to the amino acid sequence depicted in SEQ ID NO.: 2, preferably at least 90%, more preferably at least 95%, further preferably at least 98%.
As the flavanone 3-hydroxylase (F3H) gene, the dihydroflavonol 4-reductase (DFR) gene and the anthocyanidin synthetic enzyme (ANS) gene, novel or conventional genes may be used. Specifically, the F3H gene may, for example, be a gene registered on GenBank (accession number: DQ394303, AY221246, AJ493133, AY641730, AF184270, AB078956, AB201760, AY669324, AF036093, AB211958, AB187027 or AB234905). The DFR gene may, for example, be a gene registered on GenBank (accession number: AAB62873, AAC17843, AAD49343, AAQ83576, AAU93766, AAY32600, AAY32601, AAY32602, BAB40789 or BAE79202). The ANS gene may, for example, be a gene registered on GenBank (accession number: AY585677, AY228485, AF015885, AY581048, U82432, AY695817, AB208689, AY997840, AY382828, AY256380, AF026058, Y07955, AF384050, AB097216, AB087206, AB198869, AB044091, AY123768 or AB234906). The DFR gene is particularly preferably a gene derived from Gerbera which is shown by accession number: Z17221 or a gene derived from Torenia which is shown by accession number: AB012924. Further, a novel gene which is found in the present invention such as the F3H gene (SEQ ID No: 50) which is derived from moth orchid, the DFR gene (SEQ ID No: 64) or the ANS gene (SEQ ID No: 74) may be used.
Further, the present invention relates to a recombinant vector containing such a gene, particularly an expression vector.
Depending on the host species into which the gene is introduced, the expression vector has an expression regulation region such as a promoter, a terminator, a DNA replication origin, etc. The promoter may, for example, be one which induces a gene expression in petal cells, and a promoter derived from a CHS (chalcone synthase) gene of moth orchid or a promoter derived from cauliflower mosaic virus (CaMV) 35S may be mentioned. Further, the terminator may, for example, be a terminator derived from a CHS gene of moth orchid or a terminator derived from cauliflower mosaic virus (CaMV) 35S.
Specifically, it is preferred that the F3′5′H gene of Commelina communis is connected to a 3′ downstream of the promoter sequence, and a transcription termination sequence is added to a 3′ downstream of the F3′5′H gene.
The F3H gene, the DFR gene and/or the ANS gene can also be connected to a 3′ downstream of a promoter sequence and introduced into plants, like the F3′5′H gene.
The expression vector can be produced in accordance with a conventional method by using a restriction endonuclease, a ligase, etc. Further, transformation of hosts with the expression vector can also be carried out in accordance with a conventional method.
In the present invention, the method for expressing a gene in petal cells may, for example, be a microprojectile bombardment method, Agrobacterium-mediated transformation method, electroporation method, PEG method or virus-mediated transformation method.
FIG. 7 (pBIH-35S-CcF3′5′H) shows one example of a vector for carrying out transformation with the F3′5′H gene by the Agrobacterium-mediated transformation method. Further, the F3H, DFR and ANS genes can also be constructed on the same vector as containing the Commelina communis F3′5′H gene independently or together (FIGS. 7 and 8).
In the present invention, plants to be transformed are flowering plants, preferably Orchids. The Orchids may, for example, be moth orchid, cymbidium, dendrobyobium, dendrochilum, oncidium, odontoglossum, mitonia or cattleya. Among them, moth orchid and cymbidium are preferred. Cymbidium is a plant which belongs to genus Cymbidium, and moth orchid includes genus Phalaenopsis and genus Doritaenopsis. 
EXAMPLES
Now, the present invention will be described in further detail with reference to Examples. However these Examples are simply given to describe the present invention and as references of embodiments. These examples are to describe specific embodiments of the present invention and are not to restrict or limit the scope of the present invention. Further, it should be understood that various modes based on the technical concept of the present specification may be possible.
Further, general methods required for recombination of gene such as cutting and connecting DNA, transformation of E. coli, base sequence determination of a gene and PCR were basically carried out in accordance with manuals of commercially available reagents or apparatus used for each operation or experimental manual (such as “Molecular Cloning: A Laboratory Manual (Third Edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press”). For PCR, GeneAmp PCR system 9700 (PE Applied Biosystems) was used. Unless otherwise specified, an apparatus was operated in accordance with a standard operation method described in a manual attached to the apparatus. Unless otherwise specified, all examples were carried out or may be carried out by using standard techniques, and such techniques are well known and common to those skills in the art.
As a homology searching program for examining an amino acid sequence encoded with the novel gene of the present invention and known amino acid sequences, BLASTP 2.2.15 (http://www.ddbj.nig.acjp/search/blast-j.html, Document: Altschul et al., Nucleic Acids Res. (1997) 25: 3389-3402) was used. Here, homology is the degree of similarity of amino acids over the entire sequence, and its value is obtained by aligning an amino acid sequence encoded with the novel gene and known amino acid sequences in order of the high similarity, and dividing the number of the homologous amino acids between them by the number of amino acids of the compared region. Here, high similarity is an alignment result output under a state (with gap, expected value 10, with filter) that the above BLASTP program parameter was defaulted. The homology described in the present Examples is one described with respect to a known amino acid sequence which showed the highest homology in the above homology analysis.
Example 1
Transfecting a Petal of Moth Orchid with a Gene
Unless otherwise specified in Examples of the present specification, a petal of moth orchid was transfected with each gene by the following gene transfection method, and its property was evaluated. All genes had a DNA structure having a promoter at 5′ side and a terminator at 3′ side and were introduced into a petal cell in the form so as to express in the cell.
A bud of moth orchid was sterilized with a 1 wt % of sodium hypochlorite aqueous solution for five minutes and washed with sterilized water 3 times. Then, the bud was resolved into a lateral sepal, dorsal sepal and petal, and the lateral sepal, dorsal sepal and petal were left on an agar medium containing New Dogashima Medium salt (Tokuhara and Mii, Plant Cell Reports (1993) 13: 7-11., hereinafter referred to as NDM salt) and 0.6 wt % of agarose. Here, in the case of Phal. amabilis, a bud having a length of about 15 mm was used, and in the case of Dtps. Queen Beer ‘Mantenkou’, a bud having a length of about 8 mm was used.
DNA to be introduced was purified by using Hi Speed Plasmid Midi Kit (QIAGEN), and the gene was introduced by the microprojectile bombardment method. Further, in a case where plural genes were introduced simultaneously, these DNA solutions were equally mixed one another, and such a mixture was used as a DNA solution for transfection.
At that time, the adsorption of DNA on gold particles was carried out at the following ratio. DNA dissolved in 20 μl of Tris/EDTA buffer (10 mM Tris-HCL, 1 mM EDTA, pH8.0) (Each 2 μg DNA of plasmid containing a gene was mixed and dissolved in the Tris/EDTA buffer.) was mixed with 50 μl of a gold particles suspension (the particle size: 1.0 μm, 60 mg/ml of 50% glycerol), and 50 μl of 2.5 M calcium chloride and 10 μl of 0.2 M spermidine were added to 70 μl of the mixture and suspended, whereby DNA was adsorbed on gold particles. Then, a supernatant was removed by centrifugal separation, and the particles were washed with 70% ethanol and 100% ethanol respectively. Then, 60 μl of 100% of ethanol was added to an obtained precipitate to prepare a suspension. The suspension was used as a sample solution, and 10 μl of the sample solution was used for one time of gene transfection. As a gene gun, IDERA GIE-III (TANAKA Co., Ltd.) was used. The gene transfection was carried out under a condition of the distance from a nozzle to a sample of 12 cm, under a reduced pressure of −80 kPa, a helium gas pressure of 0.3 MPa and spraying time of 0.025 second.
The petal after the gene transfection was left on an NDM salt agar medium and cultured under a light-dark cycle (light intensity: 23 μmol/m2/s, light period: 16 hours, dark period: 8 hours) at 25° C.
Example 2
Searching and Cloning Commelina communis F3′5′H Gene (CcF3′5′H)
From a petal of a bud of the blue colored Asiatic dayflower (Commelina communis), all RNA was extracted by using RNeasy Plant Mini Kit (QIAGEN), and by using the RNA as a template, cDNA was prepared by using GeneRacer kit (Invitrogen).
Then, RT-PCR was carried out by using this cDNA as a template. Primers used for the PCR reaction were 35FH-1 (5′-ATGGTIGTIGARYTIATGAC-3′; SEQ ID No.: 3) and 35FH-4 (5′-CCRAAIGGIATIARYTCRAA-3′; SEQ ID NO.: 4) which were designed from a sequence of a conventionally known F3′5′H gene (GenBank accession No.: D14590, AJ011862, AB262585, D14589, AB078514, AY566988, D85184, AY275430, AF313490, AY675558, AB012925). In the reaction, a step of 98° C. for 10 seconds, 56° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template and a primer 35FH-2 (5′-TGGATGGAYYTICARGGIAT-3′; SEQ ID NO.: 5) and a primer 35FH-3(5′-CCDATIGCCCADATRTTIAC-3′; SEQ ID NO.: 6). In the reaction, a step of 98° C. for 10 seconds, 56° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles. An obtained reaction product was subjected to terminal blunting treatment and then cloning to HincII site of pUC18 (Takara Bio Inc.) to obtain p35FH23, and a partial DNA sequence contained in the p35FH23 was determined (CcF3′5′H partial sequence).
From the CcF3′5′H partial sequence, sequences of the 3′ downstream side and 5′ upstream side were analyzed by the RACE method.
The 3′RACE method was carried out by using a primer which can be designed from the CcF3′5′H partial sequence, the above Commelina communis RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were C35FH-3 (5′-ATCTCCCTCGTATCGCAACC-3′; SEQ ID NO.: 7) and GeneRacer 3′ primer. In the reaction, a step of 98° C. for 10 seconds, 58° C. for 30 seconds and 72° C. for one minute was repeated 40 cycles. Nested PCR was carried out by using an obtained reaction solution as a template, a primer C35FH-4 (5′-GAAGCTTGTGAAGCCAATGG-3′; SEQ ID NO.: 8) and GeneRacer 3′ Nested primer. In the reaction, a step of 98° C. for 10 seconds, 58° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles. An obtained reaction product was subjected to termination blunting treatment and then cloning to HincII site of pUC18 (Takara Bio Inc.) to obtain p35FHC43′, and a DNA sequence of a 3′ downstream side contained in the p35FHC43′ was determined (CcF3′5′H 3′RACE sequence).
The 5′RACE method was carried out by using a primer which can be designed from the CcF3′5′H partial sequence, the above Commelina communis RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were C35FH-6 (5′-CGTCGCCTCGTGCTCTCGCAGTATC-3′; SEQ ID NO.: 9) and GeneRacer 5′ primer. In the reaction, a step of 98° C. for 10 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 30 cycles. Nested PCR was carried out by using an obtained reaction solution as a template, a primer C35FH-5 (5′-TCTTCGAGAGCACCTTATCGAACCTC-3′; SEQ ID NO.: 10) and GeneRacer 5′ Nested primer. In the reaction, a step of 98° C. for 10 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 30 cycles. An obtained reaction products was subjected to cloning to pCR4/TOPO10 (Invitrogen) to obtain p35FH5′5, and a DNA sequence of a 5′ upstream side contained in the p35FH5′5 was determined (CcF3′5′H 5′RACE sequence).
The entire Commelina communis F3′5′H gene (CcF3′5′H) was subjected to cloning based on the CcF3′5′H 3′RACE sequence and the CcF3′5′H 5′RACE sequence. RT-PCR was carried out by using the above Commelina communis RNA as a template, a primer C35FH-7(5′-GAAAACCAATACAAAAACATACC-3′; SEQ ID NO.: 11), a primer C35FH-10 (5′-ATTGCTTCAAGTTCCCTAGC-3′; SEQ ID NO.: 12) and Ready-To-Go RT-PCR Beads (Amersham Biosciences). In the reaction, one step of 94° C. for 30 seconds, 54° C. for 30 seconds and 72° C. for 2 minutes was repeated 30 cycles. Further, semi Nested PCR was carried out by using an obtained reaction solution as a template, a primer C35FH-7 (SEQ ID NO.: 11) and C35FH-9 (5′-GTTCCCTAGCCCCGTACCAC-3′; SEQ ID NO.: 13). In the reaction, a step of 98° C. for 10 seconds, 54° C. for 30 seconds and 72° C. for 1 minute was repeated 30 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO10 (Invitrogen) to obtain p35FH79. Then, a DNA sequence of the entire Asiatic dayflower F3′5′H gene contained in the p35FH79 was determined (CcF3′5′H; SEQ ID NO.: 1). Further, the sequence of the gene of the present invention found in Commelina communis is a novel gene. The amino acid sequence encoded by the base sequence has 61% homology to the amino acid sequence (GenBank accession No.: AY856345) encoded by the F3′5′H gene of delphinium by the homology analysis.
Example 3
Preparation of an Expression Vector (pBS-P35T35) for Gene Transfection
pBS-P35T35 is a plasmid having a cauliflower mosaic virus 35S promoter (Hohn et al., Curent Topics in Microbiology and Immunology (1982) 96: 194-236), an omega sequence of tobacco mosaic virus (Gallie et al., Nucleic Acids Research (1987) 15: 3257-3273), a restriction endonuclease SwaI site and a cauliflower mosaic virus 35S terminator in this order in pBluescriptIISK-(Stratagene) (FIG. 2). A plasmid having the substantially same function as this pBS-P35T35 can be constructed as follows.
An oligonucleotide SAS-S (5′-CTAGCTAGCGGCGCGCCTGCAGGATATCATTTAAATCCCGGG-3′; SEQ ID NO.: 14) and an oligonucleotide SAS-AS (5′-CCCGGGATTTAAATGATATCCTGCAGGCGCGCCGCTAGCTAG-3′; SEQ ID NO.: 15) were denatured and then gradually cooled to room temperature. An obtained one was subjected to NheI treatment and connected to XbaI-EcoRV site of pBluescriptIISK-(Stratagene) to prepare pBS-SAS which is a plasmid DNA of which a restriction enzyme site is modified. A region amplified by PCR using a cauliflower mosaic virus genome DNA (GenBank accession V00140) as a template, a primer T-CaMV35S-SseI-F (5′-AACCTGCAGGAAATCACCAGTCTCTCTCTA-3′; SEQ ID NO.: 16) and a primer T-CaMV35S-AscI-R (5′-GGCGCGCCATCGATAAGGGGTTATTAG-3′; SEQ ID NO.: 17) was treated with a restriction endonuclease Sse8387I and AscI. This fragment was connected to Sse83871-AscI site of pBS-SAS to prepare pBS-T35S. Based on pJD301 (Leon et al., Plant Physiology (1991) 95: 968-972), a sequence of cauliflower mosaic virus 35S promoter which is cut by HindIII and HincII and an omega sequence of tobacco mosaic virus were connected to HindIII-SmaI site of pBS-T35S to prepare an expression vector (pBS-P35T35).
Example 4
Subcloning of the Commelina communis F3′5′H Gene to an Expression Vector
An Open Reading Frame part of the Commelina communis F3′5′H gene was amplified by PCR using a plasmid DNA (p35FH79) containing the above CcF3′5′H entire sequence as a template and was subjected to subcloning to an expression vector pBS-P35T35. The PCR was carried out by using a primer CcF35H-F (5′-ATGGTACCCCTTACGTACCTT-3′; SEQ ID NO.: 18), a primer CcF35H-R (5′-TTATGTTGTTTTTATATTCTTATAAACG-3′; SEQ ID NO.: 19) and p35FH79 as a template. In the reaction, a step of 94° C. for 30 seconds, 52° C. for 30 seconds and 72° C. for 1 minute and 20 seconds was repeated 25 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain p35 CcF3′5′H. The p35 CcF3′5′H is DNA to express the Commelina communis F3′5′H gene in plant cells.
Example 5
Confirmation of Expression of the Commelina communis F3′5′H Gene
A petal of a red moth orchid (Dtps. Queen Beer ‘Mantenkou’) was transfected with 1.2 μg of the p35 CcF3′5′H by the method of Example 1 and cultured for 5 days, whereby many deep purple cells emerged at the petal. On the other hand, in a case where cells were transfected with gold particles containing no such a gene, the above phenomena was not observed.
Thus, it is considered that the isolated Commelina communis F3′5′H gene imparted a flavonoid 3′5′-hydroxylase activity to the petal cell of the moth orchid, and as a result, delphinidin which is a blue pigment was produced.
Example 6
Evaluation of Enzyme Activity of the Commelina communis F3′5′H Gene
The enzyme activity of the Commelina communis F3′S′H gene was evaluated by the amount of delphinidin which is a blue pigment and compared to an already known gene (Petunia gene) which is considered to be useful for producing blue carnation.
(1) Isolation of the Petunia F3′5′H Gene (PetF3′S′H)
All RNA was isolated from a petal of a bud of commercially available Petunia (hybrid) before blossom by using RNeasy Plant Mini Kit (QIAGEN). cDNA was prepared by using this RNA as a template and superscriptII First-Strand Synthesis System (Invitrogen). Then, RT-PCR was carried out by using this cDNA as a template. As primers for the PCR reaction, PetF3′5′H1-F (5′-ATGATGCTACTTACTGAGCTTGGTG-3′; SEQ ID NO.: 20) and PetF3′5′H1-R (5′-CAACATGCGCAATTATAGCA-3′; SEQ ID NO.: 21) which were designed from two sequences (GenBank accession No.: A29011, A29013) of an already known petunia F3′5′H gene, or PetF3′5′H2-F(5′-ATGGTGCTACTTAGTGAGCTTGC-3′; SEQ ID NO.: 22) and PetF3′5′H2-R (5′-AACCAACGTAAAGGCATGTT-3′; SEQ ID NO.: 23) were used. In both cases, the reaction was carried out by repeating a step of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 2.5 minutes 45 cycles. Each obtained reaction product was subjected to cloning at SwaI site of pBS-P35T35 to obtain p35PetF3′5′H1 and p35PetF3′5′H2.
(2) Transfection with Commelina communis F3′S′H Gene and Petunia F3′5′H Gene
A petal of a red moth orchid (Dtps. Queen Beer ‘Mantenkou’) was transfected with 1.3 μg of either the p35 CcF3′5′H gene derived from Commelina communis or a gene (p35PetF3′5′H1 or p35PetF3′5′H2) derived from Petunia by the method of Example 1. Further, at the time of gene transfection, in order to measure efficiency of gene transfection, the petal was cotransfected with 0.17 μg of p35luc (Firefly Luciferase gene) as the internal standard. This p35luc was prepared by subcloning the blunt-ended Luciferase gene fragment, obtained by cutting pSP-luc+(Promega) with BgIII-XbaI, into the blunt-ended pBI221 (CLONTECH) cut with BamHI and SacI.
The gene transfected petal was static cultured for 5 days, and then grinded in liquid nitrogen and suspended in 200 μl of 0.1×Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, Promega) to prepare a sample. The Luciferase activity of the sample was measured, and the quantitative analysis of anthocyanidin was carried out.
Measurement of the Luciferase Activity
5 μl of 5×Passive Lysis Buffer (Promega) and 100 μl of Luciferase Assay Substrate (Promega) were added to 20 μl of the above sample, and then the Luciferase activity was measured by using luminometer flash'n glow LB955 (BERTHOLD TECHNOLOGIES) under a condition of measuring time of 10 seconds.
Quantitative Analysis of Delphinidin
400 μl of 2N hydrochloric acid was added to 150 μl of the above sample, and then hydrolysis treatment was carried out at 98° C. for 2 hours, and extraction with 200 μl of isoamylalcohol was carried out. The amount of delphinidin in the organic layer was measured by a liquid chromatography method under the following condition.
Apparatus: Waters2690 (Waters),
Column: Nucleosil 100-5C18 4.6×250 mm (GL. Sciences),
Column temperature: 40° C.,
Elution condition: Gradient (B liquid 20%→85% for 40 minutes, B liquid 85% for 5 minutes) was applied by using 1.5% of phosphoric acid solution (A liquid) and 1.5% phosphoric acid•20% acetic acid•25% acetonitrile aqueous solution (B liquid), and elution was carried out at a flow rate of 1 ml/minute.
Detection wavelength: 531 nm.
The amount of delphinidin was measured by an absolute analytical curve method using the standard (delphinidin hydrochloride). Further, the obtained amount of delphinidin was corrected by using Luciferase activity. Results are shown in Table 1. It is evident from Table 1 that the accumulated amount of delphinidin by the Commelina communis F3′5′H gene was higher by about five times than that of the Petunia F3′5′H2 gene. Further, the delphinidin was not detected from the sample using the Petunia F3′5′H1 gene.










TABLE 1









CcF3′5′H
PetF3′5′H2














Amount of


Amount of





delphinidin
Luc assay

delphinidin
Luc assay



(pg/sample)
(RLU/sample)
Pg/RLU
(pg/sample)
(RLU/sample)
Pg/RLU

















1
12400
265681
0.0467
4400
491562
0.0090


2
26200
743973
0.0352
13000
1063867
0.0122


3
50600
880990
0.0574
7400
757137
0.0098


4
37000
741444
0.0499
3200
530822
0.0060


Average


0.0473


0.0092


SD


0.0092


0.0026









Example 7
Transfection of a Petal of the White Moth Orchid with a F3′5′H Gene
In order to produce a blue moth orchid, a petal of white moth orchid (Phal. amabilis) was transfected with 1.6 μg of either a gene (p35 CcF3′5′H) derived from Commelina communis or a gene (p35PetF3′5′H1, p35PetF3′5′H2) derived from Petunia, like Example 6(2), in accordance with the method of Example 1. However, though the petal was transfected with such a gene, clear color change of the petal was not observed.
Therefore, a gene encoding the group of enzymes at the upstream of the F3′5′H in the anthocyanidin synthetic pathway (FIG. 1) ((1) Chalcone synthase: CHS, (2) Chalcone isomerase: CHI and (3) flavanone 3-hydroxylase: F3H) was isolated for transfection.
Example 8
Isolation of the Moth Orchid CHS Gene (PhCHS3)
All RNA was isolated from a petal just before blossom of moth orchid (Dtps. Sogo Vivien×Dtps. Sogo Yenlin) by using RNeasy Plant Mini Kit (QIAGEN). cDNA was prepared by using this RNA as a template and SuperscriptII First-Strand Synthesis System (Invitrogen). Then, RT-PCR was carried out by using this cDNA as a template. As primers for the PCR reaction, PhCHS3 F1 (5′-AAGCTTGTGAGAGACGACGGA-3′; SEQ ID NO.: 24) and PhCHS3 R1 (5′-TGGCCCTAATCCTTCAAATT-3′; SEQ ID NO.: 25) which were designed from the known moth orchid CHS gene (PhCHS) sequence (GenBank accession No.: DQ089652) were used. The reaction was carried out by repeating a step of 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 1 minute 25 cycles. A reaction product was amplified by using this reaction solution as a template under the same condition again. An obtained reaction product was subjected to cloning at SwaI site of pBS-P35T35 to obtain p35PhCHS3. Then, a DNA sequence of the entire moth orchid CHS gene contained in p35PhCHS3 was determined (PhCHS3; SEQ ID NO.: 26). The p35PhCHS3 is DNA for expressing the moth orchid CHS gene in plant cells.
Example 9
Isolation of Moth Orchid CHI Gene (PhCHI1)
All RNA was isolated from a petal just before blossom of moth orchid (Dtps. Sogo Vivien×Dtps. Sogo Yenlin) by using RNeasy Plant Mini Kit (QIAGEN). cDNA was prepared by using this RNA as a template and SuperscriptII First-Strand Synthesis System (Invitrogen).
Then, RT-PCR was carried out by using this cDNA as a template. Various plants' CHI genes have been reported (GenBank accession No.: AY700850, AY086088, DQ160231, AJ004902, AF474923, XM—470129, U03433, AB187026). As primers for the PCR reaction, CHI-dgF1 (5′-TTYCTCGSYGGBGCMGGYGWVMGVGG-3′; SEQ ID NO.: 28) and CHI-dgR1 (5′-CMGGIGAIACVSCRTKYTYICCRATVAT-3′; SEQ ID NO.: 29) which were designed from the conventionally known CHI gene were used. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles.
Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer CHI-dgF3(5′-TMIKYWCMGGISMITTYGARAARYT-3′; SEQ ID NO.: 30) and a primer CHI-dgR3 (5′-TYICCRATVATIGWHTCCARIAYBGC-3′; SEQ ID NO.:31). In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning at pCR4-TOPO (Invitrogen) to obtain PhCHIfrag 16, and a partial DNA sequence contained in the PhCHIfrag 16 was determined (PhCHI partial sequence).
From the PhCHI partial sequence, sequences of the 3′ downstream side and 5′ upstream side were analyzed by the RACE method.
The 3′RACE method was carried out by using a primer which can be designed from the PhCHI partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhCHI-GSP F1 (5′-ATGCTGCTGCCATTAACGGGTCA-3′; SEQ ID NO.: 32) and GeneRacer 3′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhCHI-GSP F2 (5′-TCCGAGAAGGTCTCCGGGAACT-3′; SEQ ID NO.: 33) and GeneRacer 3′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4-TOPO (Invitrogen) to obtain PhCHI3′RACE23. A DNA sequence of the 3′ downstream side contained in the PhCHI3′RACE23 were determined (PhCHI3′RACE sequence).
The 5′RACE method was carried out by using a primer which can be designed from the PhCHI partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhCHI-GSP R1 (5′-GCATTCGTCAGCTTCTTGCTCTCT-3′; SEQ ID NO.: 34) and GeneRacer 5′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhCHI-GSP R2 (5′-ATCACATCAGTCTCAGCCACA-3′; SEQ ID NO.: 35) and GeneRacer 5′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4-TOPO (Invitrogen) to obtain PhCHI5′RACE54. A DNA sequences of the 5′ upstream side contained in the PhCHI5′RACE54 was determined (PhCHI5′RACE sequence).
The entire of the moth orchid CHI gene (PhCHI) was subjected to cloning based on the PhCHI3′RACE sequence and the PhCHI5′RACE sequence. PCR was carried out by using the above cDNA, a primer PhCHI init (5′-ATGGCAGAAACAGTGGCGACGCCCA-3′; SEQ ID NO.: 36) and a primer PhCHI term (5′-TCAAACGACTCCATCTTGCTC-3′; SEQ ID NO.: 37). In the reaction, a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 45 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain p35PhCHI1. Then, a base sequence of the entire moth orchid CHI gene contained in the p35PhCHI1 was determined (PhCHI1, SEQ ID NO.: 38). Further, the present sequence of the gene found in moth orchid is a novel gene. The amino acid sequence encoded by the DNA sequence has 54% homology to the amino acid sequence (GenBank accession No.: DQ120521) encoded by the CHI gene of tea plant by the homology analysis. The p35PhCHI1 is DNA for expressing the moth orchid CHI gene in plant cells.
Example 10
Isolation of Moth Orchid F3H Gene (PhF3H1)
All RNA was isolated from a petal just before blossom of moth orchid (Dtps. Sogo Vivien×Dtps. Sogo Yenlin) by using RNeasy Plant Mini Kit (QIAGEN). cDNA was prepared by using this RNA as a template and SuperscriptII First-Strand Synthesis System (Invitrogen).
Then, RT-PCR was carried out by using this cDNA as a template. Various plants' F3H genes have been reported (GenBank accession No.: D0394303, AY221246, AJ493133, AY641730, AF184270, AB078956, AB078956, AB201760, AY669324, AF036093, AB211958, AB187027, AB234905). As primers for the PCR reaction, a primer F3H-dgF1 (5′-TIVGIGAYGARGABGARMGBCClAA-3′; SEQ ID NO.: 40) and a primer F3H-dgR1 (5′-ACBGCYYGRTGRTCHGCRTTCTTRAA-3′; SEQ ID NO.: 41) which were designed from the conventionally known F3H gene were used. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds, and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer F3H-dgF3 (5′-AARYTBRGKTTYGAYATGWCHGGIG-3′; SEQ ID NO.: 42) and a primer F3H-dgR3 (5′-GGHWSRACVGTDATCCAIGWBTT-3′; SEQ ID NO.:43). In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4-TOPO (Invitrogen) to obtain PhF3Hfrag 26, and a partial DNA sequence contained in the PhF3Hfrag 26 was determined (PhF3H partial sequence).
Sequences of the 3′ downstream side and 5′ upstream side from the PhF3H partial sequence were analyzed by the RACE method.
The 3′RACE method was carried out by using a primer which can be designed from the PhF3H partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhF3H-GSP F1 (5′-TTCTCATACCCAATCGGGAG-3′; SEQ ID NO.: 44) and GeneRacer 3′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, and a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhF3H-GSP F2 (5′-AATCGGGAGCCGCGATTACT-3′; SEQ ID NO.: 45) and GeneRacer 3′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4-TOPO (Invitrogen) to obtain PhF3H3′RACE33. A DNA sequence of the 3′ downstream side contained in the PhF3H3′RACE33 was determined (PhF3H 3′RACEsequence).
The 5′RACE method was carried out by using an oligonucleotide which can be designed from the PhF3H partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhF3H-GSPR1 (5′-TCTGTGTGGCGCTTCAGGCC-3′; SEQ ID NO.: 46) and GeneRacer 5′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, and a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution, PhF3H-GSP R2 (5′-TGAGGTCCGGTTGCGGGCATTTT-3′; SEQ ID NO.: 47) and GeneRacer 5′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4-TOPO (Invitrogen) to obtain PhF3H5′RACE86. A DNA sequence of the 5′ upstream side contained in the PhF3H5′RACE86 was determined (PhF3H 5′RACEsequence).
The entire of the moth orchid F3H gene was subjected to cloning based on the PhF3H 3′RACE sequence and the PhF3H 5′RACE sequence. PCR was carried out by using the above cDNA, a primer PhF3H init. (5′-ATGGCCCCAATACCATTCCTACCGA-3′; SEQ ID NO.: 48) and a primer PhF3H term. (5′-CCTTAAGCTAAAATCTCATTTAATGCCTTTGCTCC-3′; SEQ ID NO.: 49).
In the reaction, a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1.5 minute was repeated 40 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain p35PhF3H1. Then, a DNA sequence of the entire moth orchid F3H gene contained in the p35PhF3H1 was determined (PhF3H1; SEQ ID NO.: 50). Further, the sequence of the gene found in moth orchid is a novel gene. The amino acid sequence encoded by the DNA sequence has 86% homology to the amino acid sequence (GenBank accession No.: X89199) encoded by the F3H gene of Bromheadia finlaysoniana. The p35PhF3H1 is DNA for expressing the moth orchid F3H gene in plant cells.
Example 11
Transfection of a Petal of White Moth Orchid with the F3′5′H Gene and Anthocyanin Related Genes
A petal of a white moth orchid (Phal. amabilis) was cotransfected with the CHS gene (p35PhCHS3: Example 8), CHI gene (p35PhCHI1: Example 9) and F3H gene (p35PhF3H1: Example 10) which were derived from moth orchid and the F3′5′H gene (p35 CcF3′5′H: Example 2) which was derived from Commelina communis in accordance with the method of Example 1. However, the color change of the petal was not observed.
Therefore, genes encoding enzymes ((1) dihydroflavonol 4-reductase: DFR and (2) anthocyanidin synthase: ANS) at downstream of the F3′5′H gene in the anthocyanin synthetic pathway were isolated for transfection.
Example 12
Isolation of Moth Orchid DFR Gene (PhDFR)
All RNA was isolated from a petal in a bud of moth orchid (Dtps. Queen Beer ‘Mantenkou’) by using RNeasy Plant Mini Kit (QIAGEN), and cDNA was prepared by using this RNA as a template and GeneRacer kit (Invitrogen).
Then, RT-PCR was carried out by using this cDNA as a template. Various plants' DFR genes have been reported (GenBank accession No.: AAB62873, AAC17843, AAD49343, AAQ83576, AAU93766, AAY32600, AAY32601, AAY32602, BAB40789, BAE79202). Primers used for the PCR reaction were DFRD-F1(5′-TTYCAYGTIGCIACNCCNATG-3′; SEQ ID NO.: 52) and DFRD-R1(5′-DATNGCRTCRTCRAACATYTC-3′; SEQ ID NO.: 53) which were designed from the sequence of the above DFR gene. In the reaction, a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles. Nested PCR was carried out by using an obtained reaction solution as a template, a primer DFRD-F2 (5′-ATGAAYTTYCARWSIRARGAYCC-3′; SEQ ID NO.: 54) and a primer DFRD-R2 (5′-RCAIATRTAICKNCIRTTNGC-3′; SEQ ID NO.:55). In the reaction, a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 1 minute was repeated 40 cycles. Further, Nested PCR was carried out again by using an obtained reaction solution as a template, a primer DFRD-F3 (5′-GARAAYGARGTNATHAARCC-3′; SEQ ID NO.: 56) and a primer DFRD-R3 (5′-RTCRTCIARRTGNACRAAYTG-3′; SEQ ID NO.:57). In the reaction, a step of 94° C. for 30 seconds, 48° C. for 30 seconds and 72° C. for 30 seconds was repeated 40 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO (Invitrogen) to obtain PhDFR-D/pCR4, and a partial DNA sequence contained in the PhDFR-D/pCR4 was determined (PhDFR partial sequence).
From the PhDFR partial sequence, sequences of the 3′ downstream side and 5′ upstream side were analyzed by the RACE method.
The 3′RACE method was carried out by using a primer which can be designed from the PhDFR partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used for the PCR reaction were PhDFR-F1 (5′-GGTCATGCAAAAGGTCGGGCAGCGTAA-3′; SEQ ID NO.: 58) and GeneRacer 3′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhDFR-F2 (5′-GTGATCTTCACATCTTCCGCAGGAACAGT-3′; SEQ ID NO.: 59) and GeneRacer 3′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO10 (Invitrogen) to obtain PhDFR3′RACE/pCR4, and a DNA sequence of 3′ downstream side contained in the PhDFR3′RACE/pCR4 was determined (PhDFR3′RACE sequence).
The 5′RACE method was carried out by using a primer which can be designed from the PhDFR partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhDFR-R4 (5′-ATGATTCATTAAAAATCCGAAAAAAAGACCACTACAA-3′; SEQ ID NO.: 60) and GeneRacer 5′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1 minute was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhDFR-R3 (5′-AACCATGCATAATAAAGCAGATGTGTAAAT-3′; SEQ ID NO.: 61) and GeneRacer 5′ Nested primer. In the reaction, a step of 94° C. for 30 seconds, 65° C. for 30 seconds and 72° C. for 1 minute was repeated 25 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO10 (Invitrogen) to obtain PhDFR 5′RACE/pCR4, and an upstream side DNA sequence contained in the PhDFR 5′RACE/pCR4 was determined (PhDFR 5′RACE sequence).
The entire moth orchid DFR gene was subjected to cloning based on the PhDFR3′RACE sequence and the PhDFR 5′RACE sequence. RT-PCR was carried out by using the above RNA, a primer PhDFR-F8A5 (5′-AAAAAATGGAGGATGTGAGGAAGGGTCCTGTT-3′; SEQ ID NO.: 62), a primer PhDFR-R5 (5′-ACATGATTCATTAAAAATCCGAAAAAAAGACCA-3′; SEQ ID NO.: 63) and Ready-To-Go You Prime First Strand Beads (Amersham Biosciences). In the reaction, a step of 98° C. for 30 seconds, 68° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 35 cycles. An obtained reaction product was subjected to cloning to pBS-P35T35 to obtain p35PhDFR. Then, the entire DNA sequence of the DFR gene contained in the p35PhDFR was determined (PhDFR; SEQ ID NO.: 64). Further, the sequence of the gene found in moth orchid is a novel gene. The amino acid sequence encoded by the DNA sequence has 86% homology to the amino acid sequence (GenBank accession No.: AF007096) encoded by the DFR gene of Bromheadia finlaysoniana by the homology analysis. The p35PhDFR is DNA for expressing the moth orchid DFR gene in plant cells.
Example 13
Isolation of Moth Orchid ANS Gene (PhANS1)
All RNA was isolated from a petal in a bud just before blossom of moth orchid (Dtps. Queen Beer ‘Mantenkou’) by using RNeasy Plant Mini Kit (QIAGEN), and cDNA was prepared by using this RNA as a template and SuperscriptII First-Strand Synthesis System (Invitrogen).
Then, RT-PCR was carried out by using this cDNA as a template. Primers used for the PCR reaction were ANS-dgF2 (5′-TICARGGBTAYGGIAGYARRYTIGCIRMYA-3′; SEQ ID NO.: 66) and ANS-dgR2 (5′-GGYTCRCARAAIAYIRCCCAIGADA-3′; SEQ ID NO.: 67) which were designed from a known ANS gene. In the reaction, a step of 94° C. for 30 seconds, 60° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 40 cycles. By using an obtained reaction solution as a template and the same primers again, a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute was repeated 30 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO10 (Invitrogen) to obtain PhANSfrag10, and a partial DNA sequence contained in the PhANSfrag10 was determined (PhANS partial sequence). From the PhANS partial sequence, sequences of the 3′ downstream side and 5′ upstream side were analyzed by the RACE method.
The 3′RACE method was carried out by using a primer which can be designed from the PhANS partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used for the PCR reaction were PhANS3RACEGSP1 (5′-GCCCACACCGACGTCAGCTCCCTCTCCT-3′; SEQ ID NO.: 68) and GeneRacer 3′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1.5 minutes was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 70° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 25 cycles. Further, Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhANS3RACEGSP2 (5′-CGTCGGGGATGCGCTCGAGATCCTCAGC-3′; SEQ ID NO.: 69) and GeneRacer 3′ Nested primer. In the reaction, a step of 94° C. for 10 seconds, 58° C. for 10 seconds and 72° C. for 1 minute was repeated 35 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO (Invitrogen) to obtain PhANS3′RACE37, and a DNA sequence of 3′ down stream side contained in the PhANS3′RACE37 was determined (PhANS3′RACE sequence).
The 5′RACE method was carried out by using a primer which can be designed from the PhANS partial sequence, the above RNA and GeneRacer kit (Invitrogen). The primers used in the PCR reaction were PhANS5RACEGSP1 (5′-AGTCCGCGGGTTCAGTCGGCCAGATGGT-3′; SEQ ID NO.: 70) and GeneRacer 5′ primer. In the reaction, a step of 94° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 5 cycles, a step of 94° C. for 30 seconds and 70° C. for 1.5 minute was repeated 5 cycles, and then a step of 94° C. for 30 seconds, 70° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 25 cycles. Nested PCR was carried out by using an obtained reaction solution as a template, a primer PhANS5RACEGSP2 (5′-CCGTCTTCTCCGGCGGGTAGACGAGGTG-3′; SEQ ID NO.: 71) and GeneRacer 5′ Nested primer. In the reaction, a step of 94° C. for 10 seconds, 58° C. for 10 seconds and 72° C. for 1 minute was repeated 35 cycles. An obtained reaction product was subjected to cloning to pCR4/TOPO (Invitrogen) to obtain PhANS5′RACE15, and an upstream side DNA sequence contained in the PhANS5′RACE15 was determined (PhANS 5′RACE sequence).
The entire moth orchid ANS gene was subjected to cloning based on the PhANS 3′RACE sequence and the PhANS 5′RACE sequence. PCR was carried out by using the above cDNA, a primer PhANS init (5′-ATGGCCACCAAAGCAATCCCACC-3′; SEQ ID NO.: 72), and a primer PhANS term (5′-TCAATCCACAGGCGCCTTCT-3′; SEQ ID NO.: 73). In the reaction, a step of 94° C. for 30 seconds, 69° C. for 30 seconds and 72° C. for 1.5 minutes was repeated 35 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain p35PhANS1. Then, the entire base sequence of the ANS gene (PhANS1) contained in the p35PhANS1 was determined (PhANS1; SEQ ID NO.: 74). Further, the sequence of the present gene found in moth orchid is a novel gene. The amino acid sequence encoded by the DNA sequence has 58% homology to the amino acid sequence (GenBank accession No.: EF079869) encoded by the ANS gene of Anthurium by the homology analysis. The p35PhANS1 is DNA for expressing the moth orchid ANS gene in plant cells.
Example 14
Transfection of a Petal of White Moth Orchid with the F3′5′H Gene and an Anthocyanidin Related Gene
A petal of a white moth orchid (Phal. amabilis) was cotransfected with the F3′5′H gene which was derived from Commelina communis and the CHS (p35PhCHS3), CHI (35PhCHI), F3H (p35PhF3H1), DFR (PhDFR) and AnS (PhANS1) genes which were derived from moth orchid in accordance with the method of Example 1. As a result, light blue cells were observed at the petal.
Therefore, it is evident that in order to change flower color of a white moth orchid to blue, the DFR gene and ANS gene are important. Thus, in order to change flower color to deeper blue, other plants' DFR genes and ANS genes were studied.
Example 15
Isolation and the DFR Gene and ANS Gene Gerbera and Cotransfection Therewith
(1) Isolation of the Gerbera DFR Gene (GerDFR)
All RNA was isolated from a petal of a bud of commercially available Gerbera (hybrid) by using RNeasy Plant Mini Kit (QIAGEN), and cDNA was prepared by using this RNA as a template and SuperscriptII First-Strand Synthesis System (Invitrogen). Then, RT-PCR was carried out by using this cDNA as a template. As primers for the PCR reaction, GerDFR-F (5′-ATGGAAGAGGATTCTCCGGC-3′; SEQ ID NO.: 76) and GerDFR-R (5′-CTATTGGCCTTCTTTTGAACAACAAA-3′; SEQ ID NO.: 77) which were designed from a sequence (GenBank accession No.: Z17221) of a known Gerbera DFR gene (GerDFR) were used. The reaction was carried out by repeating a step of 98° C. for 10 seconds, 55° C. for 10 seconds and 72° C. for 1 minute and 30 seconds 45 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain a Gerbera DFR gene (p35GerDFR). The p35GerDFR is DNA for expressing the Gerbera DFR gene in plant cells.
(2) Isolation of Gerbera ANS Gene (GerANS)
RT-PCR was carried out by using the above cDNA as a template. Primers used for the PCR reaction were GerANS-F (5′-ATGGTGATTCAAGCAACCACA-3′; SEQ ID NO.: 78) and GerANS-R (5′-CTAGTTTTGCATCACTTCGTCTTTAT-3′; SEQ ID NO.: 79) which were designed from a sequence (GenBank accession No.: AY997842) of a known Gerbera ANS gene (GerANS).
In the reaction, a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain Gerbera ANS gene (p35GerANS). The p35GerANS is DNA for expressing the Gerbera ANS gene in plant cells.
(3) Cotransfection with the Gerbera DFR Gene and the GerANS Gene
A petal of a white moth orchid (Phal. amabilis) was cotransfected with the respective genes of the CHS (PhCHS3), CHI (PhCHI1) and F3H (PhF3H1) which were derived from moth orchid, the F3′S′H gene (CcF3′5′H) which was derived from Commelina communis and the DFR gene (GerDFR) and ANS gene (GerANS) which were derived from Gerbera. As a result, deeper blue-purple newly emerged at the petal, as compared to Example 14.
Example 16
Isolation of Torenia DFR Gene and ANS Gene and Cotransfection Therewith
(1) Isolation of Torenia DFR Gene (TorDFR)
All RNA was isolated from a petal in a bud of a conventionally available Torenia (Torenia fournieri) by using RNeasy Plant Mini Kit (QIAGEN), and cDNA was prepared by using SuperscriptII First-Strand Synthesis System (Invitrogen). RT-PCR was carried out by using this cDNA as a template. Primers used for the PCR reaction were an oligonucleotide TorDFR-F (5′-ATGAGCATGGAAGTAGTAGTACCA-3′; SEQ ID NO.: 80) and TorDFR-R (5′-CTATTCTATCTTATGTTCTCCATGG-3′; SEQ ID NO.: 81) which were designed from a sequence (GenBank accession AB012924) of an already known Torenia DFR gene (TorDFR). In the reaction, a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain a Torenia DFR gene (p35TorDFR). The p35TorDFR is DNA for expressing the Torenia DFR gene in plant cells.
(2) Isolation of Torenia ANS Gene (TorANS)
RT-PCR was carried out by using the above cDNA as a template. Primers used for the PCR reaction were TorANS-F (5′-ATGGTTTCTCCAGCATCTCCGA-3′; SEQ ID NO.: 82) and TorANS-R (5′-TCACTCAACACTCTTATCATCATGCTC-3′; SEQ ID NO.: 83) which were designed from a sequence (GenBank accession AB044091) of a known Torenia ANS gene (TorANS). In the reaction, a step of 94° C. for 30 seconds, 56° C. for 30 seconds and 72° C. for 1 minute and 10 seconds was repeated 45 cycles. An obtained reaction product was subjected to cloning to SwaI site of pBS-P35T35 to obtain a Torenia ANS gene (p35TorANS). The p35TorANS is DNA for expressing ANS gene in plant cells.
(3) Cotransfection of Torenia DFR Gene and Torenia ANS Gene
A petal of a white moth orchid (Phal. amabilis) was cotransfected with respective genes of CHS (PhCHS3), CHI (PhCHI1) and F3H (PhF3H1) which were derived from moth orchid, the F3′5′H gene (CcF3′5′H) which was derived from Commelina communis and the DFR gene (TorDFR) and ANS gene (TorANS) which were derived from Torenia in accordance with the method of Example 1, and as a result, cells having a deeper blue purple than that of Example 14 newly emerged at the petal.
Examination of the Best DFR Gene and ANS Gene
With respect to the changing white moth orchid to blue color, different genes derived from plants were introduced in order to find the best DFR gene and ANS gene, and such genes were compared and examined.
Example 17
Observation of Coloration of the Petal Cell
The coloration of the petal cells was observed by stereomicroscope SZX12 (OLYMPUS Corporation) and macroscopically observed. The standard of the degree of the coloration of the petal was that one which could be macroscopically observed was judged as “III”, one which could be observed by a stereomicroscope with a magnification of at most 32 times was judged as “II”, and one which could not be observed by a stereomicroscope with a magnification of at least 32 times was judged as “I”, and one wherein coloration was not observed as judged as “-”.
Example 18
Comparison of the ANS Genes in the Petal of the White Moth Orchid
A petal of a white moth orchid (Phal. amabilis) was transfected with one of the following three sets of genes wherein only an ANS gene varied, and the degree of coloration was observed.
(1) Moth orchid ANS gene (PhANS1)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Gerbera DFR gene (GerDFR)
(2) Gerbera ANS gene (GerANS)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Gerbera DFR gene (GerDFR)
(3) Torenia ANS gene (TorANS)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Gerbera DFR gene (GerDFR)
As a result, blue purple cells emerges on respective samples. The degrees of coloration of blue purple cells which emerged at twenty two pieces of the petal were compared. Results are shown on FIG. 3.
Example 19
Comparison of the DFR Genes in the Petal of the White Moth Orchid
A petal of a white moth orchid (Phal. amabilis) was transfected with one of the following three sets of genes wherein only a DFR gene varied, and the degree of coloration was observed.
(1) Moth orchid DFR gene (Ph DFR)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Torenea ANS gene (TorANS)
(2) Gerbera DFR gene (Ger DFR)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communisF3′5′H gene (CcF3′5′H)+Torenea ANS gene (TorANS)
(3) Torenia DFR gene (TorDFR)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Torenea ANS gene (TorANS)
As a result, blue purple cells emerges on respective samples. The degree of coloration of blue purple cells which emerged at twenty three pieces of the petal was compared. Results are shown on FIG. 4. When the PhDFR gene was used, the degree of blue-purple coloration was low, as compared to the case where the GerDFR gene or the TorDFR gene was used. It is considered that in order to produce deep blue-purple flower color, the Gerbera DFR gene (Ger DFR) and the Torenia DFR gene (TorDFR) are superior to the endogenous moth orchid DFR gene (PhDFR).
Example 20
Performance of the Commelina communis F3′5′H Gene
In order to measure the performance of the Commelina communis F3′5′H gene on the white moth orchid, comparative tests with the petunia gene was carried out.
A petal of a white moth orchid (Phal. amabilis) was cotransfected with the Commelina communis F3′5′H gene ((1) CcF3′5′H) or a petunia F3′5′H gene ((2) PetF3′5′H1, or (3) PetF3′5′H2), together with the CHS gene (PhCHS3), CHI gene (PhCHI1) and F3H gene (PhF3H1) which were derived from moth orchid and the DFR gene (GerDFR) and ANS gene (GerANS) which were derived from Gerbera. 
As a result, in a case where the petunia F3′5′H gene (PetF3′5′H1) or the petunia F3′5′H2 gene (PetF3′5′H2) was used, blue coloration was little. On the other hand, in a case where the Commelina communis F3′5′H gene (CcF3′5′H) was used, clear coloration was observed (FIG. 5).
Example 21
Necessity of Gene Transfection with F3H, CHI, CHS Genes
Necessity of respective genes in the blue color change of white moth orchids was examined. A petal of a white moth orchid (Phal. amabilis) as a material was transfected with the following four types of gene sets, and the degree of coloration was observed.
(1) Commelina communis F3′5′H gene (CcF3′5′H)+Torenia DFR gene (TorDFR)+Torenia ANS gene (TorANS)
(2) Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Torenia DFR gene (TorDFR)+Torenia ANS gene (TorANS)
(3) Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Torenia DFR gene (TorDFR)+Torenia ANS gene (TorANS)
(4) Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Commelina communis F3′5′H gene (CcF3′5′H)+Torenia DFR gene (TorDFR)+Torenia ANS Gene (TorANS)
A petal of Phal. amabilis was transfected with these genes, and the coloration of cells was observed. As a result, except (1), blue purple cells emerged. The degrees of coloration of 18 pieces of respective samples were compared, and results are shown in FIG. 6. From the results, it is evident that the F3H gene (PhF3H1) is essential for coloration.
Example 22
Analysis of Pigments Emerged by Transfecting a Petal of White Moth Orchids
In the above experiment, whether pigments contained in the blue purple cells which emerged at the petal of the white moth orchid (Phal. amabilis) was a delphinidin related pigment or not was examined. A petal of white moth orchid (Phal. amabilis) is cotransfected with the CHI gene, F3H gene and ANS gene which were derived from moth orchid, the DFR gene which was derived from Gerbera and the F3′5′H gene which was derived from Commelina communis, the petal was cultured for 0.5 days, and anthocyanidin was extracted from the petal in the same manner as in Example 6 and analyzed. As a result, delphinidin was detected as the main component of the pigment. The amount of the detected delphinidin was 16 ng per one sample (average of five samples). On the other hand, in the case of the group wherein the Commelina communis F3′5′H gene was excluded from the above gene sets, delphinidin was not detected. From the above result, it is evident that blue purple cells observed at the gene transfection of the white moth orchid were colored by a delphinidin derivative.
Example 23
Application to White Moth Orchid Other than Phal. Amabilis 
A petal of a bud having a length of 1.7 cm of a pure white large moth orchid (Phal. White Star) was cotransfected with the CHS gene, CHI gene and F3H gene which were derived from moth orchid, the DFR gene and ANS gene which were derived from Gerbera and the F3′5′H gene which was derived from Commelina communis in accordance with the method of Example 1, and new blue purple cells emerged at the petal.
Example 24
Preparation of DNA for Moth Orchid Transformation by the Agrobacterium Method
Transformation of moth orchids can be carried out by the Agrobacterium method, as well as the microprojectile bombardment method (Belamino and Mii, Plant Cell Reports (2000) 19:435-442., Mishiba et al., Plant Cell Reports (2005) 24: 297-303). DNA for transformation to obtain gene recombinant plants by these methods was constructed. Maps and preparation procedures of respective plasmids are shown on FIGS. 7 and 8.
(1) Construction of pBI-SAS1
A short fragment obtained by cutting pBS-SAS of Example 04 with NotI and HindIII was subjected to subcloning to a part that pBI-RHL described on PCT/JP02/12268 was cut with NotI and HindIII to construct pBI-SAS1. The pBI-SAS1 is a plasmid which imparts hygromycin resistance to plants by transformation with Agrobacterium. 
(2) pBS-35S-FT construction
Since a period of at least one year is required for blossom of moth orchids, DNA for expressing flowering gene FT (Kobayashi et al., Science (1999) 286: 1960-1962.) in moth orchids was constructed.
FTcDNA was prepared by amplifying all RNA prepared from whole plant of Arabidopsis by RT-PCR. For the PCR, AtFT 2nd-F (5′-GAAACCACCTGTTTGTTCAAGA-3′; SEQ ID NO.: 84) and AtFT 2nd-R (5′-TCAATTGGTTATAAAGGAAGAAGC-3′; SEQ: 85) were used as primer, a purified FT cDNA was inserted to a part formed by cutting pBS-P35T35 with SwaI, clone into which cDNA was inserted to a sense direction was selected to construct pBS-P35S-FT. The pBS-35S-FT is a plasmid of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator.
(3) Construction of pBIH-35S-CcF3′5′H
The p35 CcF3′5′H was cut with BamHI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with AscI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted to a part formed by cutting pBI-SAS1 with AscI and SwaI to construct pBIH-35S-CcF3′5′H. The pBIH-35S-CcF3′5′H is a binary vector having a T-DNA region wherein HPT which is a selection marker and CcF3′5′H of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in this order.
(4) pBS-35S-mPhCHS3 Construction
A nucleotide substitution without an amino acid substitution was introduced into a SphI cut part in the PhCHS3 cDNA to prepare a cDNA which could not be cut with SphI. PhCHS3-1038F (5′-GTAACATGTCGAGCGCTTGCGTTCTTTTCATACTCG-3′; SEQ ID NO.: 86) and PhCHS3-1073R (5′-CGAGTATGAAAAGAACGCAAGCGCTCGACATGTTAC-3′; SEQ ID NO.: 87) were used as primers for the nucleotide substitution, and cDNA was synthesized by using the pBS-35S-PhCHS3 as a template and Pyrobest (Takara Bio Inc.). Then, the template plasmid was digested by DpnI treatment to construct pBS-35S-mPhCHS3.
(5) Construction of pBS-35S-UP1
After cutting p35PhCHI1 with AscI, and a cohesive end was blunted with Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting p35PhF3H1 with XbaI, blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-UP1. The pBS-35S-UP1 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of PhCHI1 and PhF3H1.
(6) Construction of pBS-35S-Del1
The p35TorANS was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with KpnI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting p35TorDFR with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with KpnI to construct pBS-35S-Del1. The pBS-35S-Del1 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of TorANS and TorDFR.
(7) Construction of pBS-35S-Del2
The p35 CcF3′5′H was cut with AscI, a cohesive end was blunted with a Klenow fragment, and a DNA fragment was cutout by cutting the plasmid with SphI. This DNA fragment was inserted into a part formed by cutting the pBS-35S-Del1 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del2. The pBS-35S-Del2 is a plasmid in which respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator in the order of CcF3′5′H, TorANS and TorDFR.
(8) Construction of pBS-35S-Del8
The pBS-35S-UP1 was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-Del2 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del8. The pBS-35S-Del8 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of PhCHI1, PhF3H1, CcF3′5′H, TorANS and TorDFR.
(9) Construction of pBS-35S-Del9
The pBS-35S-mPhCHS3 was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-Del8 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del9. The pBS-35S-Del9 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of mPhCHS3, PhCHI1, PhF3H1, CcF3′5′H, TorANS and TorDFR.
(10) Construction of pBS-35S-Del15
The p35 CcF3′5′H was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-FT with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del15. The pBS-35S-Del15 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of CcF3′5′H and FT.
(11) Construction of pBS-35S-Del16
The p35 TorDFR was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting p35-PhANS1 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del16. The pBS-35S-Del16 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of TorDFR and PhANS1.
(12) Construction of pBS-35S-UP4
The pBS-35S-Del16 was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-UP1 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-UP4. The pBS-35S-UP4 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of TorDFR, PhANS1, PhCHI1 and PhF3H1.
(13) Construction of pBS-35S-Del17
The p35 CcF3′5′H was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-UP4 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del17. The pBS-35S-Del17 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of CcF3′5′H, TorDFR, PhANS1, PhCHI1 and PhF3H1.
(14) Construction of pBS-35S-Del18
The pBS-35S-Del15 was cut with AscI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with SphI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting the pBS-35S-UP4 with XbaI, and blunting a cohesive end with a Klenow fragment, followed by cutting with SphI to construct pBS-35S-Del18. The pBS-35S-Del18 is a plasmid wherein respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of CcF3′5′H, FT, TorDFR, PhANS1, PhCHI1 and PhF3H1.
(15) Construction of pBIH-35S-Del8
The pBS-35S-Del8 was cut with SphI, a cohesive end was blunted with a Klenow fragment, followed by cutting with AscI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting pBI-SAS1 with AscI and SwaI, to construct pBIH-35S-Del8. The pBIH-35S-Del8 is a binary vector having a T-DNA region wherein HPT which is a selection marker and respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of PhCHI1, PhF3H1, CcF3′5′H, TorANS and TorDFR.
(16) Construction of pBIH-35S-Del9
The pBS-35S-Del9 was cut with SphI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with AscI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting pBI-SAS1 with AscI and SwaI, to construct pBIH-35S-Del9. The pBIH-35S-Del9 is a binary vector having a T-DNA region wherein HPT which is a selection marker and respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of mPhCHS3, PhCHI1, PhF3H1, CcF3′5′H, TorANS and TorDFR.
(17) Construction of pBIH-35S-Del17
The pBS-35S-Del17 was cut with SphI, and a cohesive end was blunted with a Klenow fragment, followed by cutting with AscI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting pBI-SAS1 with AscI and SwaI, to construct pBIH-35S-Del17. The pBIH-35S-Del17 is a binary vector having a T-DNA region wherein HPT which is a selection marker and respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of CcF3′5′H, TorDFR, PhANS1, PhCHI1 and PhF3H1.
(18) Construction of pBIH-35S-Del18
The pBS-35S-Del18 was cut with SphI, a cohesive end was blunted with a Klenow fragment, followed by cutting with AscI to cut out a DNA fragment from the plasmid. This DNA fragment was inserted into a part formed by cutting pBI-SAS1 with AscI and SwaI to construct pBIH-35S-Del18. The pBIH-35S-Del18 is a binary vector having a T-DNA region wherein HPT which is a selection marker and respective cDNA of which transcription is controlled by CaMV 35S promoter and CaMV 35S terminator are connected in the order of CcF3′5′H, FT, TorDFR, PhANS1, PhCHI1 and PhF3H1.
Example 25
Production of Transformed Moth Orchid
A transformed moth orchid produced by using DNA (pBIH-35S-CcF3′5′H, pBIH-35S-Del8, pBIH-35S-Del9, pBIH-35S-Del17 and pBIH-35S-Del18) which was constructed as binary vector in Example 24 and Agrobacterium EHA101 strain is selected with 50 mg/ml of hygromycin. As a result, a moth orchid wherein the genes shown in Example 24 are integrated into the chromosome could be obtained.
PLB can be induced from the obtained transformed moth orchid by using a part of plant such as flower stalk or axillary bud, and clone reproduction can be carried out. By carrying out cross-breeding using the obtained transformed moth orchid as a cross-breeding parent, it is possible to obtain a progeny having integrated genes.
Thus, by using the Commelina communis F3′5′H gene, a blue toned variety can be produced from an originally colored moth orchid, and a blue variety can be newly produced from a white moth orchid.
Example 26
Evaluation of Performance of the Commelina communis F3′5′H Gene on a Petal of Cymbidium 
Ten pieces of a petal obtained from a bud having a length of 30 mm of a white Cymbidium (Cym. Lovely angel ‘The Two Virgins’) were cotransfected with one of the following three sets of genes in accordance with the method of Example 1.
(1) Commelina communis F3′5′H gene (CcF3′5′H)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Gerbera DFR gene (GerDFR)+Gerbera ANS gene (GerANS)
(2) Petunia F3′5′H2 gene (PetF3′5′H2)+Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Gerbera DFR gene (GerDFR)+Gerbera ANS gene (GerANS)
(3) Moth orchid CHS gene (PhCHS3)+Moth orchid CHI gene (PhCHI1)+Moth orchid F3H gene (PhF3H1)+Gerbera DFR gene (GerDFR)+Gerbera ANS gene (GerANS)
A transfected petal was static cultured for 5 days, and then observed by a stereomicroscope. Deep blue-purple cells newly emerged in the petals which was transfected with the gene set (1) including the Commelina communis F3′5′H gene (CcF3′5′H). On the other hand, in the petals which was transfected with the gene set (2) including the Petunia F3′5′H2 gene (PetF3′5′H2), light purple cells were observed only a part of the petals, and in the petals which was transfected with the gene set (3) containing no F3′5′H gene, flower color change was not observed at all. Results of evaluation of the degree of coloration of cells which emerged on each petal by the method described in Example 17 are shown on FIG. 9.
INDUSTRIAL APPLICABILITY
By using the Commelina communis F3′5′H gene, in addition to orchids having blue flowers which can not be accomplished by the conventional cross breeding methods, plants having various blue flower colors can be produced, and its applicability is extremely broad.
The entire disclosure of Japanese Patent Application No. 2007-066539 filed on Mar. 15, 2007 including specification, claims, drawings and summary is incorporated herein by reference in its entirety.
SEQUENCE LISTING
SEQ ID NO: 1, Nucleotide sequence encoding Commelina communis flavonoid 3′,5′-hydroxylase
SEQ ID NO: 2, Amino acid sequence of Commelina communis flavonoid 3′,5′-hydroxylase
SEQ ID NO: 3, Primer 35FH-1
SEQ ID NO: 4, Primer 35FH-4
SEQ ID NO: 5, Primer 35FH-2
SEQ ID NO: 6, Primer 35FH-3
SEQ ID NO: 7, Primer C35FH-3
SEQ ID NO: 8, Primer C35FH-4
SEQ ID NO: 9, Primer C35FH-6
SEQ ID NO: 10, Primer C35FH-5
SEQ ID NO: 11, Primer C35FH-7
SEQ ID NO: 12, Primer C35FH-10
SEQ ID NO: 13, Primer C35FH-9
SEQ ID NO: 14, Oligonucleotide SAS-S
SEQ ID NO: 15, Oligonucleotide SAS-AS
SEQ ID NO: 16, Primer T-CaMV35S-SseI-F
SEQ ID NO: 17, Primer T-CaMV35S-AscI-R
SEQ ID NO: 18, Primer CcF35H-F
SEQ ID NO: 19, Primer CcF35H-R
SEQ ID NO: 20, Primer PetF3′5′H1-F
SEQ ID NO: 21, Primer PetF3′5′H1-R
SEQ ID NO: 22, Primer PetF3′5′H2-F
SEQ ID NO: 23, Primer PetF3′5′H2-R
SEQ ID NO: 24, Primer PhCHS3 F1
SEQ ID NO: 25, Primer PhCHS3 R1
SEQ ID NO: 26, Nucleotide sequence encoding Doritaenopsis hybrid cultivar chalcone synthase
SEQ ID NO: 27, Amino acid sequence of Doritaenopsis hybrid cultivar chalcone synthase
SEQ ID NO: 28, Primer CHI-dgF1
SEQ ID NO: 29, Primer CHI-dgR1
SEQ ID NO: 30, Primer CHI-dgF3
SEQ ID NO: 31, Primer CHI-dgR3
SEQ ID NO: 32, Primer PhCHI-GSP F1
SEQ ID NO: 33, Primer PhCHI-GSP F2
SEQ ID NO: 34, Primer PhCHI-GSP R1
SEQ ID NO: 35, Primer PhCHI-GSP R2
SEQ ID NO: 36, Primer PhCHI init
SEQ ID NO: 37, Primer PhCHI term
SEQ ID NO: 38, Nucleotide sequence encoding Doritaenopsis hybrid cultivar chalcone isomerase
SEQ ID NO: 39, Amino acid sequence of Doritaenopsis hybrid cultivar chalcone isomerase
SEQ ID NO: 40, Primer F3H-dgF1
SEQ ID NO: 41, Primer F3H-dgR1
SEQ ID NO: 42, Primer F3H-dgF3
SEQ ID NO: 43, Primer F3H-dgR3
SEQ ID NO: 44, Primer PhF3H-GSPF1
SEQ ID NO: 45, Primer PhF3H-GSPF2
SEQ ID NO: 46, Primer PhF3H-GSPR1
SEQ ID NO: 47, Primer PhF3H-GSPR2
SEQ ID NO: 48, Primer PhF3H init.
SEQ ID NO: 49, Primer PhF3H term.
SEQ ID NO: 50, Nucleotide sequence encoding Doritaenopsis hybrid cultivar flavanone 3-hydroxylase
SEQ ID NO: 51, Amino acid sequence of Doritaenopsis hybrid cultivar flavanone 3-hydroxylase
SEQ ID NO: 52, Primer DFRD-F1
SEQ ID NO: 53, Primer DFRD-R1
SEQ ID NO: 54, Primer DFRD-F2
SEQ ID NO: 55, Primer DFRD-R2
SEQ ID NO: 56, Primer DFRD-F3
SEQ ID NO: 57, Primer DFRD-R3
SEQ ID NO: 58, Primer PhDFR-F1
SEQ ID NO: 59, Primer PhDFR-F2
SEQ ID NO: 60, Primer PhDFR-R4
SEQ ID NO: 61, Primer PhDFR-R3
SEQ ID NO: 62, Primer PhDFR-F8A5
SEQ ID NO: 63, Primer PhDFR-R5
SEQ ID NO: 64, Nucleotide sequence encoding Doritaenopsis hybrid cultivar dihydroflavonol 4-reductase
SEQ ID NO: 65, Amino acid sequence of Doritaenopsis hybrid cultivar dihydroflavonol 4-reductase
SEQ ID NO: 66, Primer ANS-dgF2
SEQ ID NO: 67, Primer ANS-dgR2
SEQ ID NO: 68, Primer PhANS3RACEGSP1
SEQ ID NO: 69, Primer PhANS3RACEGSP2
SEQ ID NO: 70, Primer PhANS5RACEGSP1
SEQ ID NO: 71, Primer PhANS5RACEGSP2
SEQ ID NO: 72, Primer PhANS init
SEQ ID NO: 73, Primer PhANS term
SEQ ID NO: 74, Nucleotide sequence encoding Doritaenopsis hybrid cultivar anthocyanidin synthase
SEQ ID NO: 75, Amino acid sequence of Doritaenopsis hybrid cultivar anthocyanidin synthase
SEQ ID NO: 76, Primer GerDFR-F
SEQ ID NO: 77, Primer GerDFR-R
SEQ ID NO: 78, Primer GerANS-F
SEQ ID NO: 79, Primer GerANS-R
SEQ ID NO: 80, Primer TorDFR-F
SEQ ID NO: 81, Primer TorDFR-R
SEQ ID NO: 82, Primer TorANS-F
SEQ ID NO: 83, Primer TorANS-R
SEQ ID NO: 84, Primer AtFT 2nd-F
SEQ ID NO: 85, Primer AtFT 2nd-R
SEQ ID NO: 86, Primer PhCHS3-1038F
SEQ ID NO: 87, Primer PhCHS3-1073R

## Claims

1. An isolated gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which flavonoid 3′,5′-hydroxylase comprises the amino acid sequence depicted in SEQ ID No: 2 or an amino acid sequence having at least 98% homology to the amino acid sequence depicted in SEQ ID No: 2.

2. A vector, which contains the gene as defined in claim 1.

3. A method for producing a flower color-changed plant, which comprises transfecting a red moth orchid or a cymbidium with the gene as defined in claim 1 and expressing the gene.

4. A method for producing a flower color-changed plant, which comprises transfecting a moth orchid or a cymbidium with the gene as defined in claim 1 and a gene encoding a dihydroflavonol 4-reductase of Torenia or Gerbera and expressing the genes.

5. A method for producing an Orchid family plant having a blue flower, which comprises transfecting a moth orchid having a white flower with the gene as defined in claim 1, with a gene encoding a dihydroflavonol 4-reductase of Torenia or Gerbera, with a gene encoding a flavanone 3-hydroxylase, and with a gene encoding an anthocyanidin synthase and expressing the genes.

6. A flower color-changed plant or tissue thereof comprising the isolated gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which is produced by the method as defined in claim 3.

7. A flower color-changed plant having the gene as defined in claim 1, which is a moth orchid or a cymbidium.

8. A flower color-changed plant or tissue thereof comprising the isolated gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which is produced by the method as defined in claim 4.

9. A flower color-changed plant or a tissue thereof comprising the isolated gene encoding a flavonoid 3′,5′-hydroxylase of Commelina communis, which is produced by the method as defined in claim 5.